Topoisomerase inhibitors induce irreversible fragmentation of replicated DNA in concanavalin A stimulated splenocytes.

Etoposide, a nonintercalative antitumor drug, is known to inhibit topoisomerase II. Its effects have been tested in concanavalin A stimulated splenocytes, a system of cell proliferation in which topoisomerase II is induced. The primary effect of etoposide was a strong inhibition of DNA synthesis and the production of reversible DNA breaks, presumably associated with topoisomerase II. However, prolonged (20 h) contact with the drug resulted in a secondary fragmentation by irreversible double-strand breaks that yielded unusually small DNA fragments. Surprisingly, the same effect was obtained with novobiocin, which does not produce topoisomerase II associated DNA breaks. Moreover, long-term treatment with camptothecin, a specific inhibitor of topoisomerase I which is known to induce single-strand breaks in vitro and in vivo, also produced double-strand breaks and DNA fragmentation into small pieces. These findings suggest that prolonged treatment of proliferating splenocytes by etoposide and other topoisomerase inhibitors induced DNA fragmentation by a mechanism that does not directly involve topoisomerases.

Effect of diabetes on the rhythm of [3H]thymidine incorporation in Con-A-stimulated mice splenocytes.

The chronogram of hyperglycaemia in alloxan-induced diabetic DBA/2 mice (living under conditions standardized for light-synchronized periodicity and fed ad libitum) presented an ultradian rhythm (during spring) different from the circadian blood glucose chronogram of normal control mice. Simultaneously, the [3H]thymidine ([3H]TdR) incorporation chronogram of diabetic mouse splenocytes, stimulated or unstimulated with Concanavalin-A (Con-A), was changed and unbalanced, compared to that of normal control mice. Previous experiments showed that the [3H]TdR incorporation chronogram of stimulated or non-stimulated splenocytes of normal DBA/2 mice presented seasonal variations. They were characterized generally by an ultradian rhythm. Yet, during spring, they exhibited a circadian rhythm because one phase was advanced and superimposed on the other, the latter being typically unvarying. It seems probable that the unbalanced rhythm of [3H]TdR incorporated in diabetic mouse splenocytes, stimulated or not, was responsible for a dysfunction of that population in diabetes.

Threonine starvation of concanavalin A treated lymphocytes impairs DNA polymerase alpha activity.

When cultured in a threonine-deficient medium, concanavalin A treated guinea-pig lymphocytes do not incorporate tritiated thymidine. DNA polymerase activity is strongly affected. The addition of the missing amino acid is followed by an early increase in protein and RNA synthesis and a delayed rise in DNA polymerase alpha activity associated with the onset of DNA synthesis.

Correlation between the rhythms of blood insulin levels and the stimulation of spleen lymphocytes by concanavalin-A in mice.

An ultradian rhythm of DNA synthesis and an in-vitro response to Concanavalin-A (Con-A) stimulation has been found in splenocytes of DBA/2 male mice, living under conditions standardized for light-synchronized periodicity. It was characterized by two peaks during the nycthemeral cycle. One of them was very sharp. In spite of a constant mode of synchronization of the animals, there were seasonal variations of the rhythm. Such variations did not, however, affect the position of the sharp peak, which occurred always at the same hour. The rhythm of incorporation of [3H]-thymidine by splenocytes, whether or not stimulated by Con-A, seemed correlated with that of blood insulin levels in mice. The possible effect of the physiological increase in blood insulin during the circadian cycle has been confirmed experimentally. Thus it seems probable that insulin would be one of the internal synchronizers .

Influence of amino acid deficiency and tRNA aminoacylation on DNA synthesis and DNA polymerase activity during the secondary immune response in vitro.

DNA synthesis and DNA polymerase activity are increased when KLH-primed guinea-pig lymphocytes are restimulated in vitro with the homologous antigen. This response can be modulated by glutamine deficiency and by an inhibitor of the histidyl-tRNA synthetase.

Inhibition of in vitro immune response by factor(s) prepared from lymphoid cells of old animals.

It has been shown previously that cells from long-term calf lymphoid cell cultures (8--9 months) contain factors able to inhibit in vitro immune response. We report here that extracts prepared from lymph node cells freshly removed from old cows also contain similar factors inhibiting primary and secondary in vitro responses of mouse spleen cells against sheep red blood cells. These extracts do not affect cell viability nor stimulation with B or T mitogen (LPS or Con A). They do not affect production of antibodies by previously differentiated cells such as plasmocytomes or hybridomes. The active factor(s) appear(s) to behave as protein(s) of molecular weight about 10 000. Our results suggest that analogous "ageing" events occur either in long-term cultures of lymphocytes or in lymphoid cells of senescent animals. These phenomena would involve production or activation of specific factor(s) capable of inhibiting antibody synthesis by intervening at the level of differentiation of lymphocytes stimulated either by T-dependent or T-independent antigens.

Influence on certain amino acids on steps leading to DNA synthesis in concanavalin A-treated guinea-pig lymphocytes.

When guinea-pig lymph node cells were exposed to ConA in a culture medium lacking glutamine or cysteine, no DNA synthesis occurred. The addition of the missing acid to ConA-treated lymphocytes submitted to glutamine or cysteine starvation for 40 h allowed the synthesis of DNA to take place after a period of only 10-12 h. The synthesis of DNA is preceded by a rapid increase of 3H-uridine incorporation into RNA and of 3H-leucine incorporation into protein which occurred a few hours after addition of the missing amino acid. When cycloheximide was added to lymphocytes exposed to ConA in a glutamine or cysteine deprived medium, a relative enhancement of uridine incorporation was observed. No such effect was provoked by puromycin. These results suggest the possibility of a control system in lymphocytes similar to those described in microbial cells for amino acid control of RNA synthesis.

Reactions induced in vertebrates by invertebrate cell suspensions. I/Specific effects of sea star axial organ cells injection.

Starfish axial organ cell suspensions injected subcutaneously into irradiated mice induce a characteristic angiogenesis reaction. The size of the reaction appears to be dependent upon the number of injected starfish cells. Axial organ cells also provoke a splenomegaly positive test in 16 day old chick embryos. No significant angiogenesis nor splenomegaly were induced by heat killed axial organ cells, ovocytes or branchial digestive organ cells. These results support our hypothesis that echinoid axial organ represents an ancestral primary lymphoid organ.

Reactions induced in vertebrates by invertebrate cell suspensions. II/Non adherent axial organ cells as effector cells.

In contrast to starfish axial organ cells, coelomocytes of Asterias rubens do not induce a significant reaction of angiogenesis after subcutaneous injection into irradiated mice. Axial organ cells are separated into adhering and nonadhering cells by attachment ot plastic surface. Nonadhering cells consisting mainly of small lymphocyte-like cells appear to be effector cells in the reaction. Phytogenetic implications of these observations are discussed.